Knowledge of macromolecular structure is important for understanding and developing treatments for a diverse array of diseases. Most macromolecular structures are determined with X-ray crystallography, for which a key step is to cryogenically cool the crystals to ~100 K in order to mitigate crystal damage from the ionizing radiation. For successful cooling, the crystals are usually pretreated (cryoprotected) using methods that are mainly trial and error and often require extensive screening. Here we propose a new approach in which the optimal cryoprotection scheme is predicted based on simple measurements on the crystal of interest. The best cryoprotective solution is chosen from a list of more than 30 we have characterized such that the thermal contraction of the cryosolution correctly compensates for the inherent thermal contraction of the crystal. In Aim 1 we perform a survey of known protein crystals, picking crystals to test in subsequent aims that span a range of physical characteristics likely to have an effect on the cryo-cooling result. Aim 2 tests a specific method for predicting the optimal cryosolution internal to the crystal. Aim 3, tests a similar method to predict the optimal cryosolution external to the crystal. Finally, in Aim 4 domain analysis measurements are performed to understand the details of the cooling- induced damage as the cryosolution is optimized. The long term effects of the widespread adoption of effective predictive methods of cryoprotection optimization on the field of crystallography would be to ensure that the highest quality diffraction possible is recorded from each crystal. This would (a) improve the average quality of structures determined by X-ray crystallography and (b) make the more difficult crystallographic problems more tractable. This work will impact public health by improving the reliability of biological interpretations based on macromolecular structures, and will increase the likelihood that the structure of any particular molecule with a potential impact on health can be efficiently determined. In the long term, this would improve our understanding of the causes of, and facilitate treatments for, diseases resulting from the alteration of macromolecular structure and function by either genetic or environmental factors. PUBLIC HEALTH RELEVANCE: High resolution three-dimensional structure determination of proteins, key for understanding the molecular basis of disease, are usually carried out at low temperatures. Cooling the samples is a delicate process with a high likelihood of failure. We have developed a method designed to make the cooling process more straightforward and predictable.